Project abstract Here we request funding to purchase of a Leica TIRF microscope with an iLas2 circular TIRF laser launch. This instrument will be a critical piece of equipment in my laboratory, and will be the primary data generator for at least half of the projects ongoing in my lab. In R35GM136319, the parent grant for this supplement, my laboratory proposes to determine how WASP and WDS family proteins activate Arp2/3 complex to nucleate actin filaments. Determining the mechanism of these important regulatory proteins and how they work together to assemble functional actin networks in cell depends on our ability to visualize their activities using single molecule total internal reflection fluorescence microscopy (smTIRF). While our lab is currently equipped with a TIRF microscope, our ability to carry out multi-wavelength smTIRF experiments has been limited by our ability to select the optimal TIRF angle for each fluorophore during data collection. This has prevented us from carrying out three color smTIRF experiments in which actin and two actin regulatory proteins are labeled, each with a different fluorophore. It has also complicated our efforts to visualize with single molecule sensitivity proteins labeled with fluorescent proteins (e.g. mNeonGreen), limiting the kinds of experiments that we can do. Our current TIRF microscope also suffers from uneven illumination issues that prevent us from using the entire field of view of TIRF images and make automated data processing more challenging. Therefore, we request funds to purchase a new TIRF microscope that will eliminate these issues, thereby greatly enhancing our ability to carry out multiwavelength smTIRF experiments. We believe this instrument, a Leica TIRF microscope with a iLas2 circular TIRF illuminator, will greatly enhance our ability to accomplish the specific goals in the parent grant and will increase the impact of our research on the field.